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Journal of Biomolecular NMR

, Volume 48, Issue 1, pp 1–11 | Cite as

Structure analysis of membrane-reconstituted subunit c-ring of E. coli H+-ATP synthase by solid-state NMR

  • Yasuto Todokoro
  • Masatoshi Kobayashi
  • Takeshi Sato
  • Toru Kawakami
  • Ikuko Yumen
  • Saburo Aimoto
  • Toshimichi Fujiwara
  • Hideo Akutsu
Article

Abstract

The subunit c-ring of H+-ATP synthase (Fo c-ring) plays an essential role in the proton translocation across a membrane driven by the electrochemical potential. To understand its structure and function, we have carried out solid-state NMR analysis under magic-angle sample spinning. The uniformly [13C, 15N]-labeled Fo c from E. coli (EFo c) was reconstituted into lipid membranes as oligomers. Its high resolution two- and three-dimensional spectra were obtained, and the 13C and 15N signals were assigned. The obtained chemical shifts suggested that EFo c takes on a hairpin-type helix-loop-helix structure in membranes as in an organic solution. The results on the magnetization transfer between the EFo c and deuterated lipids indicated that Ile55, Ala62, Gly69 and F76 were lined up on the outer surface of the oligomer. This is in good agreement with the cross-linking results previously reported by Fillingame and his colleagues. This agreement reveals that the reconstituted EFo c oligomer takes on a ring structure similar to the intact one in vivo. On the other hand, analysis of the 13C nuclei distance of [3-13C]Ala24 and [4-13C]Asp61 in the Fo c-ring did not agree with the model structures proposed for the EFo c-decamer and dodecamer. Interestingly, the carboxyl group of the essential Asp61 in the membrane-embedded EFo c-ring turned out to be protonated as COOH even at neutral pH. The hydrophobic surface of the EFo c-ring carries relatively short side chains in its central region, which may allow soft and smooth interactions with the hydrocarbon chains of lipids in the liquid-crystalline state.

Keywords

Membrane protein Fo subunit c Specific isotope-labeling Lipid-protein interaction Magnetization transfer Rotational resonance 

Abbreviations

EFoc

H+-ATP synthase subunit c from E. coli

TFoc

H+-ATP synthase subunit c from thermophilic Bacillus PS3

IFoc

Na+-ATPase subunit c from Ilyobacter tartaricus

MALDI-TOF MS

Matrix-assisted laser desorption/ionization time of flight mass spectrometry

Boc

tert-Butoxycarbonyl group

ssNMR

Solid-state NMR

MAS

Magic angle sample spinning

RR

Rotational resonance

DARR

Dipole-assisted rotational resonance

RFDR

Radio frequency-driven recoupling

CODSHD

13C-NMR observation of 2H-selective 1H-depolarization

TPPM decoupling

Two-pulse phase modulation

FID

Free induction decay

Notes

Acknowledgments

We are grateful to Profs. R. H. Fillingame and M. Yoshida for providing us with the E. coli MEG119 strain transformed with plasmid pCP35 harboring the gene of subunit c from E. coli and for their encouragement. We also thank Mr. Y. Hayakawa for his help in analysis of the spectra. This work was partly supported by the Target Protein Program (HA, TF, and SA), Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Technology, Sport and Culture of Japan (HA and SA), and a WCU Grant from Korean Research Foundation funded by Korea government, MEST (HA).

Supplementary material

10858_2010_9432_MOESM1_ESM.pdf (474 kb)
Supplementary material 1 (PDF 474 kb)

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Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Yasuto Todokoro
    • 1
    • 3
  • Masatoshi Kobayashi
    • 1
    • 4
  • Takeshi Sato
    • 1
  • Toru Kawakami
    • 1
  • Ikuko Yumen
    • 1
  • Saburo Aimoto
    • 1
  • Toshimichi Fujiwara
    • 1
  • Hideo Akutsu
    • 1
    • 2
  1. 1.Institute for Protein ResearchOsaka UniversitySuitaJapan
  2. 2.Department of Biophysics and Chemical BiologySeoul National UniversityKwanak-gu, SeoulKorea
  3. 3.Yokohama City UniversityTsurumi, YokohamaJapan
  4. 4.Sumitomo Rubber Industries, Ltd.Tsutsui-cho, KobeJapan

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